Protein purification in the presence of nonionic organic polymers at elevated conductivity

ABSTRACT

A method of purifying a desired protein from a preparation includes providing the preparation in a form having less than about 5% of the chromatin residing in the original production medium, contacting the preparation with a nonionic organic polymer in an amount sufficient to cause the desired protein to precipitate or adsorb on a nonionic hydrophilic surface, and adjusting a salt concentration before or during the contacting step, the adjusting step providing a sufficient salt concentration to produce a conductivity greater than physiological conductivity.

STATEMENT OF RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/770,890, filed Feb. 28, 2013, which is incorporated herein byreference in its entirety.

BACKGROUND

Embodiments disclosed herein relate to methods for purification ofproteins, including antibodies, including IgG antibodies.

The technique of protein precipitation with non-ionic organic polymers,such as polyethylene glycol (PEG), is known. It is most often used atlow salt concentrations, in the absence of added salt, but exceptionsare known (D. Gervais et al, US Patent Application 2010/0204455 A1; K.Ingham, Arch. Biochem. Biophys. 186 (1978) 106-113; K. Yamamoto et al,Virology, 40 (1970) 734-744; S. Branston et al, Biotechnol. Progr. 28(2012) 129-136).

The technique of Steric Exclusion Chromatography is known (J. Lee et al,J. Chromatogr. A 1270 (2012) 162-170; see also PCT/SG2012/000199,incorporated herein by reference and attached as Appendix A). Itexploits nonionic hydrophilic surfaces on which retention of a desiredproduct is induced by a nonionic organic polymer such as PEG. ElevatedNaCl concentrations have been reported to increase virus bindingefficiency (Lee et al supra). Conducting the technique on fluidizedparticles in the presence of elevated concentrations of NaCl has beenreported to increase recovery and purity of IgG preparations (P. Gagnonet al, J. Chromatogr. A, 1324:171-180, (2014).

Methods for reducing the content of chromatin from cell culture harvestscontaining monoclonal antibodies have been described (H. Gan et al, J.Chromatogr. A, 1291 (2013) 33-40. Chromatin is known to include DNA andhistone proteins in stable associations known as nucleosomes. Gan et alreported that chromatin and its catabolites also form associations withantibodies that limit the efficacy of subsequent fractionation methods.They reported methods that achieved chromatin reduction of 99%, andfurther reported that such reduction improved the quality ofpurification obtained by cation exchange chromatography. A variation ofa technique reported by Gan et al has been reported by Gagnon et al(2013, supra) to improve recovery and purity of IgG fractionated bysteric exclusion chromatography on fluidized hydrophilic particles.

SUMMARY

A method of purifying a desired protein from a preparation comprisingproviding the preparation in a form having less than about 5% of thechromatin residing in the original production medium, contacting thepreparation with a nonionic organic polymer in an amount sufficient tocause the desired protein to precipitate or, adsorb on a nonionichydrophilic surface, and adjusting a salt concentration before or duringthe contacting step, the adjusting step providing a sufficient saltconcentration to produce a conductivity greater than physiologicalconductivity.

DETAILED DESCRIPTION

It has been surprisingly discovered that PEG-mediated fractionationmethods performed on an impure preparation of a desired protein that hasbeen conditioned to remove at least 95% of the chromatin, where thePEG-mediated method includes at least one stage where the desiredprotein is contacted with additional salt sufficient to produce agreater than physiological conductivity, can achieve a higher degree ofdesired-protein purification than current practice with the knownhigh-functioning method of bioaffinity chromatography.

In certain embodiments, the methods disclosed herein provide multi-stepmethods for purification of a desired antibody from an impurepreparation that has been conditioned to remove at least 95% of thechromatin, including the steps of (i) contacting the impure preparationwith a non-ionic organic polymer in an amount sufficient to cause thedesired antibody to be precipitated, in the simultaneous presence of aconcentration of salt sufficient to produce a conductivity greater thannormal physiological conductivity, (ii) optionally reducing oreliminating salt from the mixture, (iii) reducing the nonionic organicpolymer to a concentration insufficient to maintain the desired antibodyin a precipitated state, leaving highly purified antibody which may befurther purified by additional fractionation methods, if desired.

In certain embodiments, the methods disclosed herein provides multi-stepmethods for purification of a desired antibody from an impurepreparation that has been conditioned to remove at least 95% of thechromatin, including the steps of (i) contacting the impure preparationwith a non-ionic organic polymer in an amount sufficient to cause thedesired antibody to be precipitated, (ii) adding salt to a concentrationof salt sufficient to produce a conductivity greater than normalphysiological conductivity, (iii) reducing or eliminating salt from themixture, (iii) reducing the nonionic organic polymer to a concentrationinsufficient to maintain the desired antibody in a precipitated state,in the presence of at least one electropositive surface, and (iv)separating the at least one electropositive surface from thepreparation, leaving highly purified antibody which may be furtherpurified by additional fractionation methods, if desired.

In certain embodiments, the methods disclosed herein provides multi-stepmethods for purification of a desired antibody from an impurepreparation that has been conditioned to remove at least 95% of thechromatin, including the steps of (i) contacting the impure preparationwith a non-ionic organic polymer in an amount sufficient to cause thedesired antibody to be precipitated, in the simultaneous presence of aconcentration of salt sufficient to produce a conductivity greater thannormal physiological conductivity, (ii) reducing the nonionic organicpolymer to a concentration insufficient to maintain the desired antibodyin a precipitated state, leaving highly purified antibody which may befurther purified by additional fractionation methods, if desired.

In certain embodiments, the methods disclosed herein provides multi-stepmethods for purification of a desired antibody from an impurepreparation that has been conditioned to remove at least 95% of thechromatin, including the steps of (i) contacting the impure preparationwith a non-ionic organic polymer in an amount sufficient to cause thedesired antibody to be accreted on the surface of a monolith with anon-ionic hydrophilic surface, in the presence of salt sufficient toproduce a conductivity greater than normal physiological conductivity,(ii) washing the accreted antibody with a clean solution of polymer andsalt, then optionally washing the accreted antibody with a cleansolution of polymer lacking or deficient in salt, (iii) then eluting thepurified antibody by reducing the nonionic organic polymer to aconcentration insufficient to maintain the desired, antibody in anaccreted state.

In certain embodiments, the methods disclosed herein provides multi-stepmethods for purification of a desired antibody from an impurepreparation that has been conditioned to remove at least 95% of thechromatin, including the steps of (i) contacting the impure preparationwith a non-ionic organic polymer in an amount sufficient to cause thedesired antibody to be precipitated, (ii) adding salt to a concentrationof salt sufficient to produce a conductivity greater than normalphysiological conductivity, (iii) reducing or eliminating salt from themixture, (iii) reducing the nonionic organic polymer to a concentrationinsufficient to maintain the desired antibody in a precipitated state,in the presence of at least one electropositive surface, and (iv)separating the at least one electropositive surface from thepreparation, leaving highly purified antibody which may be furtherpurified by additional fractionation methods, if desired.

In any of the previous embodiments, nonionic hydrophilic particles mayoptionally be present at any stage of the method. In some suchembodiments, the nonionic hydrophilic particles may have an average sizeof 10 nanometers, or 100 nanometers, or 1 micron, or 10 microns, or 100microns, or an intermediate or larger size. In some embodiments, theparticle may be magnetic or paramagnetic, enabling their collection in amagnetic field or on a magnetic surface.

In certain embodiments, the method may be practiced with dead-endfiltration media, where the precipitate is retained while unprecipitatedcontaminants pass through, and are thereby eliminated.

In certain embodiments, the method may be practiced with centrifugation,where the precipitate is sedimented, enabling the disposal of thecontaminant-containing supernatant.

In some embodiments where the method is practiced in a centrifugalformat, an impure IgG preparation from which at least 95% or thechromatin has been removed is precipitated by the addition ofpolyethylene glycol with an average molecular weight of 6000 (PEG-6000)and NaCl to produce a precipitating solution contain 19% PEG-6000 and0.8 M NaCl. The precipitate is sedimented by centrifugation and thecontaminant-containing supernatant is discarded. The precipitated IgG isoptionally resuspended in a fresh solution of 19% PEG-6000, 0.8 M NaCland a buffering substance to confer a desired pH value, resedimented,and the supernatant discarded. The precipitate may then be optionallyresuspended in a fresh solution of PEG-6000, and a buffering substanceto confer a desired pH value, but lacking or deficient in salt; thenresedimented, after which the supernatant is discarded. The precipitatedhighly purified IgG is then resolubilized in a buffer formulated to beconsistent with any follow-on fractionation step or application of thepurified antibody that may be desired.

In some embodiments where the method is practiced in a filtrationformat, an impure IgG preparation from which at least 95% or thechromatin has been removed is precipitated by the addition ofpolyethylene glycol with an average molecular weight of 6000 (PEG-6000)and NaCl to produce a precipitating solution contain 19% PEG-6000 and0.8 M NaCl. The precipitate is captured on a membrane filter with anaverage pore size of 0.22 microns, while the contaminant-containingsupernatant flows through the membrane and is discarded. Theprecipitated IgG is optionally resuspended in a fresh solution of 19%PEG-6000, 0.8 M NaCl and a buffering substance to confer a desired pHvalue, refiltered, and the supernatant discarded. The precipitate maythen be optionally resuspended in a fresh solution of PEG-6000, and abuffering substance to confer a desired pH value, but lacking ordeficient in salt; then refiltered, after which the supernatant isdiscarded. The precipitated highly purified IgG is then resolubilized ina buffer formulated to be consistent with any, follow-on fractionationstep or application of the purified antibody that may be desired, andoptionally filtered through the same membrane previously used toseparate the precipitated antibody from soluble contaminants in thesupernatant.

In some embodiments where the method is practiced in a filtration formatand employs nonionic hydrophilic particles as nucleation centers, animpure IgG preparation from which at least 95% of the chromatin has beenremoved, starch particles amounting to 5% w/v are added before the IgGis forced onto the particles by the addition of polyethylene glycol withan average molecular weight of 6000 (PEG-6000) and NaCl to produce aprecipitating solution contain 19% PEG-6000 and 0.8 M NaCl. IgG accreteson the particles instead of forming purely-IgG precipitates. Theparticles with accreted IgG are captured on a membrane filter with anaverage pore size of 0.22 microns, while the contaminant-containingsupernatant flows through the membrane and is discarded. The particleswith accreted IgG are optionally resuspended in a fresh solution of 19%PEG-6000, 0.8 M NaCl and a buffering substance to confer a desired pHvalue, refiltered, and the supernatant discarded. The particles withaccreted IgG may then be optionally resuspended in a fresh solution ofPEG-6000, and a buffering substance to confer a desired pH value, butlacking or deficient in salt; then refiltered, after which thesupernatant is discarded. The highly purified IgG is then resolubilizedin a buffer formulated to be consistent with any follow-on fractionationstep or application of the purified antibody that may be desired, andfiltered through the membrane, which retains the starch particles whilethe purified IgG is retained by the membrane and discarded.

In some embodiments where the method is practiced in a magnetic particleformat and employs nonionic hydrophilic particles as nucleation centers,an impure IgG preparation from which at least 95% of the chromatin hasbeen removed, starch-coated magnetic nanoparticles amounting to 1% w/vare added before the IgG is forced onto the particles by the addition ofpolyethylene glycol with an average molecular weight of 6000 (PEG-6000)and NaCl to produce a precipitating solution contain 19%. PEG-6000 and0.8 M NaCl. IgG accretes on the particles instead of forming purely-IgGprecipitates. The particles with accreted IgG are captured in a magneticfield, while the contaminant-containing supernatant is discarded. Theparticles with accreted IgG are optionally resuspended in a freshsolution of 19% PEG-6000, 0.8 M NaCl and a buffering substance to confera desired pH value, recaptured in a magnetic field, and the supernatantdiscarded. The particles with accreted IgG may then be optionallyresuspended in a fresh solution of PEG-6000, and a buffering substanceto confer a desired pH value, but lacking or deficient in salt; thenrecaptured in a magnetic field, after which the supernatant isdiscarded. The highly purified IgG is then resolubilized in a bufferformulated to be consistent with any follow-on fractionation step orapplication of the purified antibody that may be desired. Thenanoparticles are removed by capturing them in a magnetic field.

In some embodiments where the method is practiced in a magnetic particleformat and employs nonionic hydrophilic particles as nucleation centers,an impure IgG preparation from which at least 95% of the chromatin hasbeen removed, starch-coated magnetic nanoparticles amounting to 1% w/vare added before the IgG is forced onto the particles by the addition ofpolyethylene glycol with an average molecular weight of 6000 (PEG-6000)and NaCl to produce a precipitating solution contain 19% PEG-6000 and0.8 M NaCl. IgG accretes on the particles instead of forming purely-IgGprecipitates. The particles with accreted IgG are captured in a magneticfield, while the contaminant-containing supernatant is discarded. Theparticles with accreted IgG are optionally resuspended in a freshsolution of 19% PEG-6000, 1.0 M NaCl, 50 mM Tris, pH 8.0, recaptured ina magnetic field, and the supernatant discarded. The highly purified IgGis then resolubilized. The nanoparticles are removed by capturing themin a magnetic field, and the IgG is applied to a column packed with ahydrophobic anion exchanger such as Capto adhere (GE Healthcare), wherethe antibody binds. The antibody is then eluted by reducing the NaClconcentration to 300 mM.

In some embodiments where the method is practiced in a fixed-bedchromatography format and employs a monolith with a nonionic hydrophilicsurface as nucleation center for accretion of IgG, an impure IgGpreparation from which at least 95% of the chromatin has been removed isforced onto the monolith surface in the presence of polyethylene glycolwith an average molecular weight of 6000 (PEG-6000) and NaCl to producea precipitating solution contain 19% PEG-6000 and 0.8 M NaCl. IgGaccretes on the monolith surface instead of producing precipitates. Thecontaminant-containing supernatant flows through the monolith and isdiscarded. The monolith is washed with a fresh solution of 19% PEG-6000,1.0 M NaCl, 50 mM Tris, pH 8.0. The highly purified IgG is eluted fromthe monolith by applying a solution of 1.0 M NaCl, 50 mM Tris, pH 8.0.In some such embodiments where it is desired to conduct additionalpurification, the IgG is optionally applied to a column packed with ahydrophobic anion exchanger such as Capto adhere (GE Healthcare), wherethe antibody binds. The antibody is then eluted by reducing the NaClconcentration to 300 mM. In a closely related embodiment, the final washbuffer contains 19% PEG-6000, 50 mM MES, pH 6.0 and the IgG is eluted in50 mM MES pH 6.0 and applied to a cation exchanger and subsequentlyeluted by increasing the salt concentration and/or by increasing the pH.In a closely related embodiment, the final wash buffer contains 19%PEG-6000, 50 mM sodium phosphate, 100 mM NaCl, pH 7.0 and the IgG iseluted in 50 mM sodium phosphate, 100 mM NaCl, pH 7.0 and applied to ahydrophobic cation exchanger for additional purification. In a closelyrelated embodiment, the final wash buffer contains 19% PEG-6000, 10 mMsodium phosphate, pH 7.0 and the IgG is eluted in 10 mM sodiumphosphate, pH 7.0 and applied to hydroxyapatite for additionalpurification. It will be recognized that all of the additionalpurification steps described above have the effect of removing residualPEG from an IgG preparation, in addition to removing additionalcontaminants that may be present.

In some embodiments, the desired protein may be an antibody. In someembodiments, the desired protein may be an IgG antibody. In someembodiments, the desired protein may be an IgM antibody. In someembodiments, the desired protein may be a monoclonal antibody. In someembodiments, the desired protein may be a non-antibody protein. In someembodiments, the desired protein may be a clotting factor. In someembodiments, the desired protein may be fibrinogen. In some embodiments,the desired protein, may be Factor VIII. In some embodiments, thedesired protein may be a complex or Factor VIII and von Willebrandfactor, where the complex is also known as anti-hemophiliac factor.

In certain embodiments, a concentration of salt sufficient to produce aconductivity greater than normal physiological conductivity may besubstantially greater than normal physiological conductivity. Wherenormal conductivity may range from 12 to 16 mS/cm, a value considered tobe substantially greater may include 20 mS/cm, or 30 mS/cm, or 40 mS/cm,or 50 mS/cm, or 60 mS/cm, or 70 mS/cm, or 80 mS/cm, or 90 mS/cm, or 100mS/cm, or 150 mS/cm, or 200 mS/cm, or an intermediate or higher value,for example up to the conductivity of a saturated solution of NaCl.Experimental data indicate the most effective contaminant reduction andIgG recovery may be obtained in a range from about 50 to 150 mS/cm,corresponding to a concentration of NaCl ranging from about 0.5 to about1.5 M. In other cases, the term greater than physiological conductivitymay be understood to be 17, or 18, or 19 mS/cm, or an intermediatevalue. It is to be understood that every IgG behaves differently and thecomponents of the impure preparation in which the IgG resides alsoimpose an influence, so it will be necessary to conduct simpleexperiments to determine the most favorable conductivity to accommodatea particular IgG preparation. It is important to realize that inaddition to the advantage of enabling PEG precipitation to achieve ahigher level of purity, the reduction of contaminants, including acidiccontaminants, also reduces the necessary capacity of the at least oneelectropositive surface, which renders it more effective and may alsopermit the dimensions of that surface to be reduced.

In certain embodiments, the salt employed to increase conductivity maybe a neutral salt, such as sodium chloride, potassium chloride, sodiumacetate, potassium acetate. In certain embodiments the salt may be achaotropic salt, such as guanidine acetate, guanidine hydrochloride,guanidine sulfate, phosphate, sodium thiocyanate, or potassiumthioscyanate, among others. In certain embodiments, the use ofprecipitating salts will be avoided, including sodium sulfate, potassiumsulfate, ammonium sulfate, sodium citrate, potassium citrate, ammoniumcitrate, potassium phosphate, because they have a strong tendency tocreate a spontaneous phase separation, producing a concentrated PEGsolution floating on top of a concentrated salt solution. For mostpurposes, and especially as a starting point, NaCl will generally be thesalt of choice.

In certain embodiments, the concentration of salt is greater than 2.0 M,or greater than 2.5 M, or greater than 3 M, or greater than 4 M, up to asaturated solution. In some such embodiments, the salt is NaCl. In somesuch embodiments, the salt is KCl.

In certain embodiments, during the process stage where the conductivityis above physiological conductivity, especially including valuessubstantially above physiological conductivity, the need to optimizeoperating pH may be reduced or suspended. At low conductivity values, pHmodulates charge interactions among proteins. When proteins are at theirisoelectric point, their net charge is zero and they are minimallyself-repellant. When pH is above their isoelectric point, they have anet negative charge, and with increasing pH become increasinglyself-repellant. When pH is below their isoelectric point, they have anet positive charge, and with decreasing pH become increasinglyself-repellant. Thus when a given IgG is at or close to its isoelectricpoint, it generally favors precipitation by PEG. Since salt ions competewith electrostatic interactions between charged bodies in solution, theyreduce the degree of repellency between proteins at pH values away fromtheir isoelectric point, which reduces or abolishes the effect of pH.The practical significance in the context of the disclosed methods isthat in some embodiments, the presence of salts at concentrations thatproduce high conductivities largely eliminates the need to adjust pH.For the purposes of customizing the method to a particular IgG in aparticular impure preparation, a neutral pH of 7.0 makes a good startingpoint, and it may be unnecessary to adjust it.

In some embodiments where a salt is present at a concentration greaterthan 2 M, the pH of the solution is 7, or 8, or 8.5, higher, or 6, or6.5, or another value between 6 and 8.5, or lower than 6.

In some such embodiments, the salt is NaCl or KCl, or a combinationthereof, or a combination of either or both with other salts.

In some embodiments, the nonionic organic polymer may be polyethyleneglycol (PEG), or polypropylene glycol, or dextran, or cellulose, orstarch, or polyvinylpyrrolidone, among others, or a combination.

In some embodiments, the nonionic organic polymer will be PEG with apolymer size of about 2,000 Daltons (D), or 3,000 D, or 4,000 D, or5,000 D, or 6,000 D, or 8,000 D, or 10,000 D, or 12,000, D, or anintermediate polymer size. Experience documents that the smaller theaverage polymer size, the higher the concentration required to achieveprecipitation. This is not simply to adjust for mass; there is aprogressive effect mediated by the fact that the effectivity of PEGpolymers is proportional to their hydrodynamic radius independent mass,so that PEG polymers of the same mass but different degrees of branchinghave different abilities to mediate precipitation. In some embodiments,a combination of polymer sizes may be employed.

In some embodiments, the PEG will be PEG-6000, and the concentration ofPEG required to achieve IgG precipitation at physiological or lowerconductivity values, or to maintain the IgG in a precipitated state atthose conductivity values, may range from 12% to 18%, or 14-16%. In someembodiments, the concentration of PEG-6000 in a concentration of saltsufficient to produce a conductivity of about 80 mS/cm, such as 1 MNaCl, will need to be elevated in order to achieve IgG precipitationand/or maintain IgG in a precipitated state. In one such embodimentwhere the concentration of PEG-6000 required to achieve precipitation atphysiological concentration is 15-16%, the concentration of PEG-6000required to achieve precipitation in the presence of 1 M NaCl is 18-22%.This reflects a known but generally overlooked effect of salt whereby itdecreases the hydrodynamic radius of the PEG. These concentrations canbe used as guidelines or starting points, with the understanding thataccommodating the unique individual characteristics of each IgG cloneand the impure preparation in which it resides will requireoptimization.

In some embodiments, the impure IgG preparation may consist of a cellculture harvest, such as from mammalian cells, yeasts, or bacteria. Insome embodiments, the impure preparation may consist of an extract ofcultured cells. In some embodiments, the impure IgG preparation mayconsist of a bodily fluid, such as plasma, serum, milk or other bodilyfluids.

In some embodiments, the impure preparation may be a cell cultureharvest that has been processed to remove cells. In one such embodiment,the harvest may be previously conditioned by physical methods such ascentrifugation and filtration.

In all of the previous embodiments, practicing the disclosed methods onan impure preparation that has been conditioned to remove at least 95%clearance of chromatin and associated catabolites disproportionatelyincreases the ability of the system to achieve high purification. Insome such embodiments, the harvest may be conditioned by contact withone or more positively charged surfaces where the positive charge isconferred by multivalent organic ions immobilized on the surface. Inanother such embodiment, the conditioning method involves contacting thedesired product preparation with soluble multivalent organic ions. Inanother such embodiment, the conditioning method involves contacting thedesired product preparation with soluble and/or insoluble multivalentorganic ions.

In some embodiments, the preparation can be reduced in chromatin to alevel at least 95% lower than in the original biological source by aconditioning method that involved contacting the harvest with one ormore multivalent organic ions. In some such embodiments, the multivalentorganic ion is electropositive.

In some embodiments, a soluble electropositive multivalent ion comprisesone selected from the group of cations consisting of methylene blue,ethacridine, chlorhexidine, benzalkonium chloride, cetyl trimethylammonium bromide, and combinations thereof.

In some embodiments, the multivalent ion is insoluble by virtue of beingcovalently attached to a surface.

In some embodiments, the insoluble electropositive multivalent ion isselected from the group consisting of a primary amino group, a secondaryamino group, a tertiary amino group, a quaternary amino group, a complexcation containing more than one positive charges conferred by one ormore types of amino groups, and combinations thereof.

In some embodiments, the insoluble electropositive multivalent ion istris(2-aminoethyl)amine (TREN).

In some embodiments, the multivalent organic ion is electronegative.

In some embodiments, the soluble electronegative multivalent ion isselected from the group consisting of heptanoic acid, heptenoic acid,octanoic acid, octenoic acid, nonanoic acid, nonenoic acid, decanoicacid, methyl blue, and combinations thereof.

In some embodiments, the insoluble electronegative multivalent ion isselected from the group consisting of a phospho group, a carboxyl group,a sulfo group, a complex anion containing more than one negative chargeconferred by one or more types of negatively charged groups, andcombinations thereof.

In some embodiments, the insoluble complex electronegative multivalentmay be iminodiacetic acid or nitriloacetic acid.

In some embodiments, the insoluble multivalent ion has a 1:1 affinityfor a metal ion.

In some embodiments, allantoin may be optionally present at asupersaturating concentration in a range selected from the groupconsisting of (a) from about 0.6% to about 50%, (b) from about 0.7% toabout 20%, (c) from about 0.8% to about 10%, (d) from about 0.9% toabout 5%, (e) from about 1% to about 2%, and an intermediate rangesthereof.

In one or more of the preceding embodiments, conditioning the impurepreparation with organic multivalent ions comprises contacting thesample with a soluble electropositive organic additive. In some suchembodiments, the electropositive organic additive comprises at least onespecies from the group consisting of ethacridine, methylene blue,cetyltrimethylammonium bromide. In some such embodiments, theconcentration of such a species, or aggregate concentration of acombination of species is in the range of 0.001 to 1%, or 0.01 to 0.1%,or 0.02 to 0.05%, or 0.01 to 0.1%, or an intermediate value. In somesuch embodiments the pH of the preparation may be adjusted up to analkaline value that does not cause significant reduction of recovery ofthe desired IgG. In one such embodiment, the pH may be adjusted up to apH value within a half pH unit of the antibody isoelectric point, ormore if experimental results indicate that antibody recovery isacceptable, but such adjustments are generally not necessary. To theextent that any pH adjustment is made, a value within 1 pH unit of theprotein isoelectric point will suffice, or within 1.5 pH units, orwithin 2 pH units or more.

In one or more of the preceding embodiments, conditioning the impurepreparation with organic multivalent ions comprises contacting thesample with a soluble electronegative organic additive. In some suchembodiments, the electronegative organic additive comprises at least onespecies from the group consisting of heptanoic acid, heptenoic acid,octanoic acid, octeneoic acid, nonanoic acid, nonenoic acid, decanoicacid, methyl blue. In some such embodiments, the concentration of such aspecies, or total concentration of a combination of species is in therange of 0.001 to 10%, or 0.01 to 1%, or 0.1 to 0.5%. In some suchembodiments the pH of the preparation may be adjusted down to an acidicvalue that does not cause significant reduction of recovery of thedesired protein. In some such embodiments, the pH of the preparation maybe adjusted to the range of 3.5 to 6.5, 4.0 to 6.0, 4.5 to 5.5, 5.0 to5.3, 5.15 to 5.25, or 5.2, or another intermediate value.

In one or more of the preceding embodiments, conditioning the impurepreparation with organic multivalent ions comprises contacting thesample with undissolved allantoin. In some such embodiments, the addedallantoin resident in an impure preparation may amount to about 0.6% to50%, or 0.7 to 20%, or 0.8 to 10%, or 0.9 to 5%, or 1 to 2%, or anintermediate value. In one or more of the preceding embodiments, theaverage particle size of the dry allantoin is selected to be thesmallest size available, with the goal of achieving the highest totalsurface area of the undissolved allantoin in a supersaturated solution.In one such embodiment, the allantoin is granulated to produce a smallerparticle size.

In some embodiments, where a multimodal organic ion immobilized on asolid surface is electropositive, the immobilized multimodal organic ionmay be a nitrogen containing group, such as a primary amino group, or asecondary amino group, or a tertiary amino group, or a quaternary aminogroup, or a combination or polymer of such groups. In some suchembodiments, the nitrogen-containing compound may be 2(aminoethyl)amine(TREN). In some embodiments, the positively charged nitrogen-containinggroup may be an imine, or a pyridine, or other electropositive group. Insome embodiments, the positive charge of a nitrogen-containing compoundmay reside on a residue other than a nitrogen atom, such as a sulfuratom.

In one or more of the preceding embodiments, conditioning the impurepreparation with organic multivalent ions comprises contacting thesample with a nonionic or zwitterionic surfactant at a concentrationlower than its critical micelle concentration.

In one or more of the preceding embodiments, conditioning of the impurepreparation with organic multivalent ions comprises (i) providing afirst component which is a first solid substrate having anelectronegative surface; (ii) contacting the impure preparation with thefirst component, wherein the operating conditions substantially preventthe binding of the desired protein to the first component; and (iii)separating the desired protein with a reduced chromatin content from thefirst component. In some such embodiments, the first electronegativesurface may be accompanied by a second electronegative surface.

In one or more of the preceding embodiments, conditioning of the impurepreparation with organic multivalent ions comprises (i) providing afirst component which is a first solid substrate having anelectropositive surface; (ii) contacting the impure preparation with thefirst component, wherein the operating conditions substantially preventthe binding of the desired protein to the first component; and (iii)separating the desired protein with a reduced chromatin content from thefirst component. In some such embodiments, the first electropositivesurface bears residues of 2(aminoethyl)amine. In some such embodiments,the first electropositive surface may be accompanied by a secondelectropositive surface.

In one or more of the preceding embodiments, conditioning of the impurepreparation with organic multivalent ions comprises (i) providing afirst component which is a first solid substrate having anelectropositive surface; (ii) providing a second component which is asecond solid substrate having an electronegative surface; (iii)contacting the impure preparation with the first and second components,wherein the first and second components are configured such that theimpure preparation may contact both components simultaneously, whereinthe operating conditions substantially prevent the binding of thedesired protein to the first or second components; and (iv) separatingthe desired protein with a reduced chromatin content from the first andsecond components. In some such embodiments, the first electropositivesurface bears residues of 2(aminoethyl)amine (TREN).

In one or more of the preceding embodiments, conditioning of the impurepreparation with organic multivalent ions comprises (i) contacting theimpure preparation with at least one solid surface comprising at leastone surface-bound ligand capable of binding a metal, wherein thesurface-bound ligand capable of binding a metal is initiallysubstantially devoid of a metal, wherein operating conditions areselected to substantially prevent the binding of the desired protein tothe at least one solid surface and (ii) separating the impurepreparation from the at least one surface-bound ligand.

In one or more of the preceding embodiments, an impure preparationalready treated with a soluble electropositive or electronegativeorganic additive and/or a solid surface bearing an electronegative,electropositive, or metal affinity ligand, may be subsequently flowedthrough a device, the fluid-contact surface of which comprises positivecharges.

In one embodiment illustrating application of a chromatin-directedclarification method, allantoin is added to a cell culture harvest in anamount of 1% (v/v). The cell culture may contain cells, or the cells maypreviously have been removed. Methylene blue is added to a concentrationof 0.025% (w/v). Alternatively, ethacridine may be added to aconcentration of 0.025%. Alternatively, 0.025% cetyl trimethyl ammoniumbromide may be added to a concentration of 0.025%. Alternatively, acombination of these or other electropositive organic additives may beused at a combined concentration of 0.025%. The mixture is thenincubated stirring for 2 hours. Particles bearing the electropositivemetal affinity ligand 2(aminoethyl)amine (TREN) are added in an amountof 2-5% v:v. The mixture is incubated stirring for 4 hours then thesolids are removed by any expedient means. The remaining solutioncontaining the desired protein may be optionally flowed through a depthfilter bearing positive charges on its fluid contact surface.

In another embodiment illustrating application of a chromatin-directedclarification method, allantoin is added to a cell culture harvest in anamount of 1% (v/v). The cell culture may contain cells, or the cells maypreviously have been removed. 0.7% heptanoic acid is added.Alternatively 0.4% octanoic acid is added. Alternatively 0.4% octenoicacid is added. Alternatively 0.3% pelargonic (nonanoic) acid is added.Alternatively 0.3% nonenoic acid is added. Alternatively 0.2% capricacid is added. Alternatively, 0.5% methyl blue is added. Alternatively,a combination of these or other electronegative organic additives may beused. The mixture is then incubated stirring for 2 hours. Particlesbearing the electropositive metal affinity ligand 2(aminoethyl)amine(TREN) are added in an amount of 2-5% v:v. The mixture is incubatedmixing for 4 hours then the solids removed by any expedient method. Theremaining solution containing the desired protein may be optionallyflowed through a depth filter bearing positive charges on its fluidcontact surface.

In some embodiments, the degree of chromatin reduction of a particularconditioning method is estimated by measuring the degree of DNAreduction, and the degree of histone reduction, and averaging the twovalues. In some such embodiments, DNA is measured by an intercalatingdye assay, while histones are typically measured by immunoassay. In somesuch embodiments, because the amount of histone is a direct function ofthe amount of chromatin, the total histone content can be measured bydetermining the content of 1 species of histone, and adjusting thequantity in proportion to the relative proportions of other histones inchromatin. For example, total histone might be estimated by measuringhistone H1, then multiplying times 9 to account for the mass ratio of H1to other histones in intact chromatin. Alternatively, total histonemight be estimated by measuring H2a, H2b, H3, or H4, and multiplying theresult from any one of them by 4.5. Alternatively, individual assaysmight be run for H1 and H2a, and H2b, and H3, and H4, and the resultsadded together. DNA assays may alternatively be aided by polymerasechain reaction technology. However, experimental data indicate thatusing DNA to estimate total chromatin may cause total chromatin to beunderestimated due to enzyme-mediated hydrolysis of the DNA followingcell death during production. Histone and DNA assays may both requirespecial extraction procedures to obtain accurate results.

In certain embodiments, the IgG preparation may be contacted with one ormore antiviral compounds during one or more stages of the method. Insome such embodiments, the one or more antiviral compounds are selectedfrom the group consisting of ethacridine, methylene blue, benzalkoniumchloride, chlorhexidine, cetyltrimethyl ammonium bromide,tri(n-butyl)phosphate.

In certain embodiments, nonionic hydrophilic particles employed duringperformance of the method are nanoparticles or microparticles. Incertain such embodiments, the nonionic hydrophilic particles are porous.In certain embodiments, the particle size is between about 50 nm andabout 500 μm, or is between about 50 nm and about 50 μm, or is betweenabout 50 nm and about 4 μm, or is between about 50 nm and about 3 μm, oris between about 50 nm and about 1 μm, or is between about 100 nm andabout 1, or is between about 200 nm and about 2 μm, or is between about200 nm and about 500 nm, or is between about 500 nm and about 1 μm, oris between about 5 μm and about 50 μm.

In some embodiments, there are provided methods of purifying a desiredprotein from a preparation comprising providing the preparation in aform having less than about 5% of the chromatin residing in the originalproduction medium, contacting the preparation with a nonionic organicpolymer in an amount sufficient to cause the desired protein toprecipitate or adsorb on a nonionic hydrophilic surface, and adjusting asalt concentration before or during the contacting step, the adjustingstep providing a sufficient salt concentration to produce a conductivitygreater than physiological conductivity.

In some embodiments, methods disclosed herein further comprise retainingthe precipitated desired protein on a porous membrane, thereby allowingsoluble contaminants to pass through the membrane.

In some embodiments, methods disclosed herein further compriseseparating the precipitated desired protein from soluble contaminants bysedimenting the precipitate by means of centrifugation.

In some embodiments, the nonionic hydrophilic surface comprises oneselected from the group consisting of a membrane, a monolith, particles,the particles being optionally magnetic, and combinations thereof.

In some embodiments, the method is carried out in a single integratedapparatus.

In some embodiments, the nonionic organic polymer is polyethylene glycol(PEG).

In some embodiments, an average polymer size of the nonionic organicpolymer is in a range selected from the group consisting of (a) fromabout 1,500 Daltons to about 15,000 Daltons, (b) from about 2,000Daltons to about 12,000 Daltons (c) from about 3,000 Daltons to about10,000 Daltons, (d) from about 4,000 Daltons to about 8,000 Daltons, and(f) from about 5,000 Daltons to about 6,000 Daltons.

In some embodiments, the conductivity is at least 1 mS/cm greater thanphysiological conductivity. In some embodiments, the conductivity isgreater than physiological conductivity by greater than about 1 mS/cm, 5mS/cm, 10 mS/cm, 20 mS/cm, 40 mS/cm, 80 mS/cm, 160 mS/cm, up to aconductivity corresponding to a saturated solution of a selectedsolution of salt or salts, or an intermediate increment. In someembodiments, a range of conductivities greater than physiological may befrom about 35 mS/cm to about greater than physiological conductivity byabout 135 mS/cm is employed.

In some embodiments, the salt is selected from the group consisting ofsodium chloride, potassium chloride, sodium acetate, potassium acetate,sodium thiocyanate, potassium thiocyanate, guanidinium hydrochloride,and combinations thereof.

In some embodiments, the salt is sodium chloride at a concentrationselected from the group consisting of (a) from about 0.5 M to about 1.5M, (b) from about 2.0 M to about 3.0 M, and intermediate ranges thereof.

In some embodiments, methods further comprise adjusting a pH to a rangeselected from the group consisting of (a) from about 5 to about 9, (b)from about 6 to about 8, (c) from about 6.5 to about 7.5, (d) from about7.5 to about 8.5, and intermediate ranges thereof.

In some embodiments, the preparation is obtained from a biologicalsource selected from the group consisting of a cell culture medium, anextract from cultured organisms, and a bodily fluid.

Terms below are defined so that the methods disclosed herein may beunderstood more readily. Additional definitions are set forth throughoutthe detailed description.

“Chromatin” refers the basic composition of chromosomes. In its intactform in living cells it dominantly comprises DNA and histone proteins,associated with smaller amounts of other proteins and peptides. It isorganized into nucleosomes, which comprise an octamer of histoneproteins including 2 each of histones 2a, 2b, 3, and 4, wrapped with1.65 turns of DNA. Nucleosomes are linked in linear areas by sections oflinker DNA, which are associated with histone H1. Chromatin begins tobreak down coincident with cell death. In cell culture process such asused to produce recombinant proteins, chromatin and its break-downproducts are expelled into the cell culture media where they may formassociations with the constituents of the cell culture media, includingthe desired recombinant product. The term “chromatin catabolites” may beused to refer to chromatin break-down product. These breakdown productsinclude arrays containing 2-30 or more nucleosomes, individualnucleosomes, nucleosome fragments, DNA, and histone proteins (Gan et alsupra, Gagnon et at (2013) supra). “Histone proteins” are understood torepresent chromatin catabolites. “DNA” regardless of its size, isunderstood to represent a species of chromatin catabolites. Individual“nucleosomes” and nucleosome arrays are understood to representchromatin catabolites.

“Steric exclusion chromatography” refers to a purification method inwhich retention of an antibody molecule (e.g., an IgG or other antibodyproduct) is mediated by simultaneous mutual steric exclusion of anonionic organic polymer such as PEG from the hydrophilic surfaces of anantibody and a nonionic particle. It is believed that no direct chemicalinteraction occurs between the protein surface and the particlessurface. This distinctively endows the method with the ability tomaintain binding over a wider range of conditions than is possible withtraditional chromatography methods such as ion exchange or hydrophobicinteraction chromatography.

“Hydrated surface” or “highly hydrated surface” or “hydrophilic surface”refers to surface that interacts strongly with water, potentiallythrough hydrogen bonding, electrostriction, or some combination of thetwo mechanisms. Such interactions may be mediated by chemical groupssuch as hydroxyls, negative charges, or positive charges, or unchargedpolar groups. The presence of hydratable chemical groups may be a basicfeature of the native composition of a given material, such as aparticle or convective chromatography material, or it may be added orenhanced by chemical modification to immobilize such groups on thesurface, including but not limited to carbohydrates and ureides.So-called hydrophobic surfaces are generally considered not to be highlyhydrated, but surfaces that include strongly hydratable groups incombination with hydrophobic residues may nevertheless be sufficientlyhydrated to practice the methods disclosed herein.

“Aggregate(s)” refers to an association of two or more molecules that isstable at physiological conditions and may remain stable over a widerange of pH and conductivity conditions. Aggregates frequently compriseat least one biomolecule such as a protein, nucleic acid, or lipid andanother molecule or metal ion. The association may occur through anytype or any combination of chemical interactions. Aggregates ofantibodies can be classified into two categories: “Homo-aggregates”refers to a stable association of two or more antibody molecules;“Hetero-aggregates” refers to a stable association of one or moreantibody molecules with one or more non-antibody molecules. Thenon-antibody component may consist of one more entities from the groupconsisting of a nucleotide, an endotoxin, a metal ion, a protein, alipid, or a cell culture media component.

“Antibody” refers to an immunoglobulin of the class IgG, IgM, IgA, IgD,or IgE derived from human or other mammalian cell lines, includingnatural, or genetically modified forms such as humanized, human,single-chain, chimeric, synthetic, recombinant, hybrid, mutated,grafted, and in vitro generated antibodies. “Antibody” may alsoinclude-composite forms including but not limited to fusion proteinscontaining an immunoglobulin moiety, or immunoconjugates created bysynthetic linkage of an IgG to another functional moiety, includinganother antibody, an enzyme, a fluorophore or other signal generatingmoiety, biotin, a drug, or other functional moiety.

“Antibody Product” refers to a proteinaceous entity at least part ofwhich comprises an antibody or a portion of an antibody. The simplestexample is an antibody. Compound examples include Fc-fusion proteins,which are functional non-antibody proteins covalently bound byrecombinant means to the Fc portion of an antibody. Another compoundexample is an antibody conjugate, or immunoconjugate, which consists ofan antibody linked to another moiety, most often by synthetic means, toincrease the functionality of the antibody, for example making itfluorescent so that it can be used in immunoassays, or binding it to anenzyme for the same purpose, or two a cytotoxin for killing cancercells, or to other moieties for other purposes. Other antibody productsare bivalent compounds (such as fusions between two antibody fragments)having two domains with binding specificities for two separate targets.

“Endotoxin” refers to a toxic heat-stable lipopolysaccharide substancepresent in the outer membrane of gram-negative bacteria that is releasedfrom the cell upon lysis. Endotoxins can be generally acidic due totheir high content of phosphate and carboxyl residues, and can be highlyhydrophobic due to the fatty acid content of the lipid-A region.Endotoxins can offer extensive opportunity for hydrogen bonding.

“Non-ionic organic polymer” refers to a naturally occurring or synthetichydrocarbon composed of linked repeating organic subunits that lackcharged groups. It may be linear, dominantly linear with some branching,or dominantly branched. Examples suitable to practice the methodsdisclosed herein include but are not limited to polyethylene glycol(PEG), polypropylene glycol, and polyvinylpyrrolidone (PVP). PEG has astructural formula HO—(CH₂—CH₂—O)_(n)—H. Examples include, but are notlimited to compositions with an average polymer molecular weight rangingfrom less than 100 to more than 10,000 daltons. The average molecularweight of commercial PEG preparations is typically indicated by ahyphenated suffix. For example, PEG-6000 refers to a preparation with anaverage molecular weight of about 6,000 daltons. The effectiveconcentration of such agents varies with the identity of the polymer andthe characteristics of the antibody product being processed by themethods disclosed herein.

“Organic multivalent ion” refers to an organic molecule, ion or salt ofnatural or synthetic origin that embodies at least one charge and atleast one additional chemical functionality, thus rendering itmultivalent. In certain embodiments, an organic multivalent ion the atleast one additional chemical functionality is an additional charge suchthat the organic multivalent ion bears two or more like or differingcharges. The organic multivalent ion may bear a net positive, netnegative, or net neutral charge. Where the organic multivalent ion isnet positive it may be provided together with anions such as chlorides,bromides, sulfates, organic acids, lactates, gluconates, and any otheranion not incompatible with the method. In certain embodiments certainof the positive charges of the organic multivalent ion are supplied byamine, imine or other nitrogen moieties. The organic multivalent ion mayadditionally be of mixed chemical character and include hydrophobicresidues, other functional moieties and/or it may possess the ability toparticipate in other types of chemical interactions including, forexample, the ability to participate in hydrogen bonds, hydrophobicinteractions, pi-pi bonding, metal coordination, and intercalation.Examples of positively charged organic multivalent ions in certainembodiments include but are not limited to the diamino acids, di-, tri,or larger homo- or hetero-peptides, such as polylysine, polyarginine,polyhistidine, polyornithine; polyethyleneimine; polyallylamine;polydimethrine, polymethylacrylamidopropyltrimethylammonia;polydiallyldimethylammonia; polyvinylbenzyltrimethylammonia;polyvinylguanidine; poly(N-ethyl-4-vinylpyridine; DEAE-dextran;DEAE-cellulose; ethacridine (CAS number 442-16-0;7-ethoxyacridine-3,9-diamine); tris(2-aminoethyl)amine; guanidine;chlorhexidine; alexidine; citricidal, protamine; spermine; spermidine;salmine; chitosan; and variants and derivatives of the foregoing. Forexample, variants and derivatives of ethacridine are understood toinclude 9-aminoacridine (aminacrine), 3,6 acridinediamine (proflavin),acrisorcin, acrizane (phenacridane), acridine orange, quinacrine,acricide, acridone, acridine-9-carboxylic acid, acranil(1-[(6-chloro-2-methoxy-9-acridinyl)amino]-3-(diethylamino)-2-propanoldihydrochloride), phenosafranin, phenoxazine, phenothiazine, acriflavine(3,6-diamino-10-methylacridinium, chloride and 3,6-acridineidiamine),and salts thereof (e.g. chlorides, bromides, sulfates, lactates,gluconates.) Another class of effective electropositive multivalentorganic ions includes thiazines, such as methylene blue, itsderivatives, analogues, and salts thereof. Where the organic multivalention is net electronegative it may be provided together with cations suchas sodium or potassium, or any other cation not incompatible with themethod. In certain embodiments certain of the negative charges of theorganic multivalent ion are supplied by carboxyl, phospho, or sulfomoieties. The organic multivalent ion may additionally be of mixedchemical character and include hydrophobic residues, other functionalmoieties and/or it may possess the ability to participate in other typesof chemical interactions including, for example, the ability toparticipate in hydrogen bonds, hydrophobic interactions, pi-pi bonding,metal coordination, and intercalation. Examples of negatively chargedorganic multivalent ions in certain embodiments include but are notlimited to the fatty acids such as heptanoic acid, heptenoic acid,octanoic acid, octenoic acid, nonanoic acid, nonenoic acid, decanoicacid, methyl blue, anionic polymers, and salts thereof (e.g. chlorides,bromides, sulfates, lactates, gluconates.)

“Polynucleotide” refers to a biopolymer composed of multiple nucleotidemonomers covalently bonded in a chain. DNA (deoxyribonucleic acid) andRNA (ribonucleic acid) are examples of polynucleotides. Polynucleotidescan have a high propensity for formation of hydrogen bonds.

“Protein” refers to any of a group of complex organic macromoleculesthat contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur andare composed principally of one or more chains of amino acids linked bypeptide bounds. The protein may be of natural or recombinant origin.Proteins may be modified with non-amino acid moieties such as throughglycosylation, pegylation, or conjugation with other chemical moieties.Examples of proteins include but are not limited to antibodies, clottingfactors, enzymes, and peptide hormones.

“Protein preparation” refers to any aqueous or mostly aqueous solutioncontaining a protein of interest, such as a cell-containing cell cultureharvest, a (substantially) cell-free cell culture supernatant, or asolution containing the protein of interest from a stage ofpurification.

“Surfactant” includes “surface active agents” such as a class of organicmolecules that generally embody a hydrophobic portion and a hydrophilicportion, causing them to be referred to as amphiphilic. At sufficientconcentrations in aqueous solutions, surfactants can self-associate intoclusters with the hydrophobic portions concentrated at the center tominimize contact with water, and the hydrophilic portions radiatingoutwards to maximize contract with water. In the presence of biologicalpreparations, especially those containing materials that have ahydrophobic character or possess areas of hydrophobic character, thehydrophobic portion of surfactants tend to associate spontaneously withsome portions of the hydrophobic material and increase their solubilitythrough the influence of the hydrophilic portion of the surfactant. Theymay also be used to modulate hydrophobic interactions that occur betweendiffering hydrophobic materials both dissolved in an aqueous solvent.Examples of surfactants suitable for practicing certain embodiments ofthe methods disclosed herein include but are not limited to nonionicsurfactants such as polysorbate surfactants (e.g., Tween 20,Polyoxyethylene (20) sorbitan monolaurate, and Tween 80, Polyoxyethylene(20) sorbitan monooleate) and Triton (e.g., polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether), and zwitterionic surfactantssuch as CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO(3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate),and octyl glucoside (e.g.,(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-octoxyoxane-3,4,5-triol).

“Synthetic particles” may range in size from less than 100 nm to morethan 100 microns. They may be porous or non-porous. They may polymeric,composed for example of polymethacrylates, polyacrylates, agarose,cellulose, dextran, or other polymers, or they may be inorganic, such assilica. They may be of uniform structure throughout, or they may becompound, consisting of an inner core of one material such as a metalalloy or hydrophobic polymer, and coated with an applied surface that ishighly hydrated or permits the attachment of chemical groups to producea highly hydrated surface. “Synthetic particles” may include particlesdesigned for chromatographic applications, or particles intended forapplications entirely distinct from the field of chromatography.

“Impure preparation” refers to any aqueous or mostly aqueous solutioncontaining a protein of interest, such as a cell-containing cell cultureharvest, a (substantially) cell-free cell culture supernatant, or a cellextract, or a bodily fluid, or a solution containing the protein ofinterest from a stage of purification.

“Ureide” refers to a cyclic or acyclic organic molecule of natural orsynthetic origin that comprises one or more urea moieties or derivativesthereof. In certain embodiments, the methods disclosed herein providesureides such as urea, uric acid, hydantoin, allantoin (CAS number97-59-6; alcloxa, aldioxa, hemocane, ureidohydantoin, 5-ureidohydantoin,glyoxylureide, glyoxylic acid diureide, 2,5-dioxo-4-imidazolidinylurea), purines, and derivatives thereof. In certain embodiments, themethods disclosed herein provides organic molecules of the formulaR—CO—NH—CO—NH₂ or R—CO—NH—CO—NH—CO—R′ or R′ R″NH—CO—NR′″R″″ where therelevant “R-groups” may be H or any organic moiety.

“Virus” or “virion” refers to an ultramicroscopic (roughly 20 to 300 nmin diameter), metabolically inert, infectious agent that replicates onlywithin the cells of living hosts, mainly bacteria, plants, and animals:composed of an RNA or DNA core, a protein coat, and, in more complextypes, a surrounding envelope.

In certain embodiments, steric exclusion chromatography (SXC) isconducted on fluidized hydrophilic nonionic particles. In certainembodiments, the SXC particles are microparticles that may range fromless than 10 microns to more than 200 microns. In certain embodimentsthe SXC particles are nanoparticles that may range in size from lessthan 10 nm to 1000 nm. In certain embodiments, the SXC particles may benon-porous. In certain embodiments, the SXC particles maybe microporous.In certain embodiments, the SXC particles may be macroporous. In certainembodiments, the SXC particles may be constituted in such a way as toenable their capture on a magnetic surface or in a magnetic field.

In certain embodiments, SXC is conducted on fluidized hydrophilicnonionic particles. In certain embodiments, the SXC particles aremicroparticles that may range from less than 10 microns to more than 200microns. In certain embodiments the SXC particles are nanoparticles thatmay range in size from less than 10 nm to 1000 nm. In certainembodiments, the SXC particles maybe non-porous. In certain embodiments,the SXC particles may be microporous. In certain embodiments, the SXCparticles may be macroporous. In certain embodiments, the SXC particlesmay be constituted in such a way as to enable their capture on amagnetic surface or in a magnetic field.

In certain embodiments, one or more salts other than NaCl may be used ina washing step. Such salts may include so-called chaotropic salts,sometimes referred to as structure-breaking salts, as exemplified bysodium or potassium isothiocyanate, or so-called kosmotropic salts,sometimes referred to as structure-forming salts, as exemplified byammonium sulfate, sodium citrate, or potassium phosphate.

In certain embodiments, washing agents other than salts may be used incombination with or instead of salt, such as nonionic chaotropes likeurea, or nonionic or zwitterionic surfactants such as CHAPS, CHAPSO,octaglucoside, Tween, or Triton; or nonionic organic solvents such asethylene glycol or propylene glycol; or sugars such as sucrose orsorbitol, or chelating agents. Due to their ionic nature and their,potential to interfere with anion exchange processes, electropositivechelating agents such as TREN may be preferred over electronegativechelating agents such as EDTA. Other washing agents may also beconsidered, such as amino acids like arginine, withelectro-positive-dominant species preferred for the same reason as withchelating agents.

In certain embodiments, the method is performed as a single unitoperation.

In certain embodiments, the PEG used to promote retention of the IgG onthe steric exclusion chromatography particles may be of an average sizeof 8 kDa, or 6, or 4, or 3, or 2, or 1 kDa. In certain embodiments, thesubstance used to promote retention of the IgG on the steric exclusion,chromatography particles may be a polymer other than PEG, for examplepolypropylene glycol, polyvinyl pyrrolidone, dextran, or anothernonionic organic polymer.

In certain embodiments, the desired antibody product is a fully intactantibody such as an IgG with a molecular mass of about 150 kDa.

In certain embodiments, the sample consists of conditioned cell culturesupernatant (CCS). In certain embodiments, the CCS is conditioned bycentrifugation, or flocculation, or filtration, or some combination ofthese techniques. In certain embodiments, the CCS is conditioned by moreinclusive means, including the use of chemical additives thatparticularly reduce chromatin content and/or aggregate content of thepreparation. In certain embodiments, the CCS is clarified by a methodthat removes 95% or more of chromatin, and coincidentally achievessubstantial removal of non-histone host protein, endotoxin, and virus,and reduction of aggregate content to 1% or less, while supportingantibody recovery of 90-99%.

In certain embodiments, the claimed methods disclosed herein reducesaggregate content. In certain embodiments, the claimed methods disclosedherein reduce the content of antibody fragments.

It will be recognized by the person experienced in the art that manyvariations of the above processes can be employed without departing fromits essential elements. For example, embodiments may involve uncouplingthe process steps so that it is conducted in two, or three, or more unitoperations.

In certain embodiments, the claimed methods disclosed herein may bepreceded by, or followed by, or both preceded and followed by, otherpurification methods. In some such embodiments, the disclosed methodsmay be particularly followed by a fractionation method whereby thedesired IgG is retained on a surface, so that residual PEG may be washedaway and the antibody subsequently eluted from the surface on which itwas retained, now free of PEG. In some such embodiments, the surface onwhich the antibody is retained may be a chromatography medium from thegroup consisting of a cation exchange chromatography medium, an apatitemedium, a mixed mode medium that combines positive charges andhydrophobicity. In one such embodiment, a mixed mode chromatographymedium that combines positive charges and hydrophobicity is representedby the commercial chromatography product marketed under the name Captoadhere (GE Healthcare).

In some embodiments, given that the IgG preparation at the end of thedisclosed method will contain a non-IgG-precipitating concentration of anonionic organic polymer, a follow on purification method may beselected specifically to reduce or eliminate that polymer from thepreparation. In one such embodiment, the IgG may be bound to at leastone electronegative surface, to which the nonionic organic polymer doesnot bind, and is thereby eliminated. In one such embodiment, the atleast one electronegative surface may be a so-called cation exchanger,such as known and marketed for the purpose of performing chromatographicfractionation of proteins, including IgG preparations. In one suchembodiment, the cation exchanger may be in the form of a membrane, amonolith, a column of packed particles, or another physical format. Inanother embodiment, an at least one electronegative surface may consistof a so-called multimodal chromatography material with a surfacecomposition that permits it to participate in hydrophobic and/orhydrogen binding interactions in addition to electrostatic interactionsthrough its electronegativity. In another embodiment, an electropositivechromatography material with a surface composition that also allows itto participate in hydrophobic interactions and hydrogen bonding may beemployed. In one such embodiment, a PEG-containing IgG preparation wasapplied to a column packed with Capto adhere at 1 M NaCl, pH 7.0. Theantibody bound, the PEG did not and was thereby eliminated. The antibodywas eluted by reducing the NaCl concentration to 0.3M, whereupon the IgGeluted.

In certain embodiments, SXC particles are added to cell culturesupernatant containing a monoclonal IgG antibody. Polyethylene glycol(PEG) is added to the level required for the IgG to be retained by theparticles. The liquid containing contaminants that are not bound to theparticles is removed, for example by filtration through a membrane, andreplaced with clean PEG buffer, hereinafter referred to as a washbuffer. Except for the presence of PEG, the buffer formulation issuitable for binding of residual contaminants to the electropositiveparticles while antibody remains unbound. The liquid is again removed,for example by filtration through a membrane. The SXC particles aresuspended in a buffer lacking or deficient in PEG but otherwise similarto the wash buffer, with the result that the IgG dissociates from theparticles in a soluble form. The IgG is collected, for example byfiltration through a membrane that retains the SXC particles andelectropositive particles, which maybe discarded or recycled.

In certain embodiments, SXC particles are added to an impure preparationcontaining a monoclonal IgG antibody. PEG is added to the level requiredfor the IgG to be retained by the particles in the presence of 0.8 MNaCl, which is also present in solution. The liquid containingcontaminants that are not bound to the particles is removed, for exampleby filtration through a membrane, and replaced with clean PEG-NaClbuffer. The high-salt wash buffer is removed, for example by filtrationthrough a membrane, and is replaced with clean PEG buffer lacking ordeficient in NaCl. The wash buffer is removed. The SXC particles aresuspended in a buffer lacking or deficient in PEG, with the result thatthe IgG dissociates from the particles in a soluble form. The IgG iscollected for example by filtration through a membrane that retains theSXC particles, which may be discarded or recycled. In some embodiments,particles for SXC are omitted from the process.

In certain embodiments, SXC particles are added to an impure preparationcontaining a monoclonal IgG antibody. PEG is added to the level requiredfor the IgG to be retained by the particles. The liquid containingcontaminants that are not bound to the particles is removed, for exampleby filtration through a membrane, and replaced with clean PEG bufferthat also contains 0.8 M NaCl. The particles are again collected on themembrane while the high-salt buffer is removed, and replaced with cleanPEG buffer lacking or deficient in NaCl. The particles are re-collectedon the membrane and the buffer is removed. The SXC particles aresuspended in a buffer lacking or deficient in PEG, with the result thatthe IgG dissociates from the particles in a soluble form. The SXCparticles are removed by the membrane, while the purified IgG flowsthrough. In some embodiments, particles for SXC are omitted from theprocess.

In any of the embodiments involving the use of membranes, it will beapparent to the person of ordinary skill in the art that they can beperformed in either a so-called dead-end filtration format, or atangential flow filtration format, where the former may be moreconvenient for conducting the disclosed methods at lab scale or todevelop the most effective buffer conditions, and where the latter maybe more appropriate for industrial scale applications, where it may bepartially or fully automated.

Additional objects and advantages of the methods disclosed herein willbe set forth in part in the description which follows, and in part willbe obvious from the description, or may be learned by practicing themethods disclosed herein. The objects and advantages of the methodsdisclosed herein will be realized and attained by means of the elementsand combinations specified in the claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the methods disclosed herein as claimed.

EXAMPLES Example 1

Definition of conditions for IgG binding to steric exclusionchromatography media. An experiment was conducted to determine the saltconcentration that supports the most effective reduction of host cellproteins from an anti-HER2 monoclonal antibody produced by mammaliancell culture. The experiment was conducted using nonionic hydrophilicstarch particles with an average diameter of about 30 microns. Theparticles were mixed with different aliquots of cell culture supernatant(CCS). The host protein concentration of the CCS was about 243,011 partsper million (ppm). The sample was treated with allantoin, ethacridine,anion exchange particles and cation exchange particles, which reducedthe host protein concentration to 165,213 ppm. PEG-6000 was added to afinal concentration of 18%. NaCl was then added to different aliquots toproduce a series containing 0.0, 0.2, 0.4, 0.8, and 1.0 M. Host cellprotein contaminant levels in the treated samples were 15,558 ppm, 1,994ppm, 662 ppm, 90 ppm, and 266 ppm. Purification at 0.8 M thus representsan improvement of 99.95%; more than a 3 log reduction.

Example 2

Determination of salt concentration for anion exchange treatment. Theantibody of example 1 was evaluated by VEAX. Samples were applied to aVEAX column equilibrated in separate experiments at different pH valuesranging from 3 to 9 but lacking salt. Samples were also applied to aVEAX column at pH 8 but including different levels of salt from 0 M to 1M. The most effective contaminant reduction was achieved at pH 8.0 inthe absence of salt. Subsequent experiments refined the target pH at8.2. Performance was inferior at both pH 8.15 and 8.25. Under theseconditions, VEAX achieved up to 99.8% reduction of host proteins,including host proteins, DNA, virus, and endotoxin. These conditionsdefined the conditions for contaminant extraction by anion exchange inconjunction with SXC, for this antibody.

Example 3

Determination of salt concentration for anion exchange treatment. Theantibody of experiments 1 and 2 was applied to anion exchange membranesin stacked flat membrane and hollow fiber formats. Contaminant reductionwas essentially the same as under the same conditions for VEAX. Theseexperimental results show that integration of SXC with anion exchangemembranes can achieve more than 6 logs of purification (99.9999%).

Example 4

Combination of SXC with electropositive particles. HER2 IgG from cellculture supernatant was bound to starch particles in the presence of 19%PEG-6000 at 1 M NaCl. Electropositive particles in the form of DowexAG1×8 200-400 mesh were also present in an amount of 4% w/v. The fluidwas removed by filtration through a membrane with 0.22 micron pores,then replaced with clean buffer containing 19% PEG-6000, 1 M NaCl, and50 mM Tris, pH 8.0. The fluid was removed and replaced with clean buffercontaining 19% PEG-6000, and 50 mM Tris, pH 8.0. This step was repeated,then the fluid removed by filtration. The fluid was replaced with 50 mMTris, pH 8.0. Host cell proteins were reduced from 142,000 parts permillion (ppm) in the original sample to about 120 ppm.

Example 5

Combination of SXC with electropositive particles. The form of example 4was repeated except that the electropositive particles were not addeduntil the starch particles were washed with 19% PEG-6000, and 50 mMTris, pH 8.0. The fluid was removed then replaced with 50 mM Tris, pH8.0 to dissociate the IgG from the SXC particles. Host cell proteinswere reduced from 142,000 parts per million (ppm) in the original sampleto about less than 1 ppm.

Example 6

Combination of SXC with electropositive particles. The form of example 5was repeated except using UNOsphere Q particles in place of Dowexparticles. Results with reduction of host cell protein were equivalent,but IgG recovery was higher.

Example 7

Combination of SXC with electropositive particles. The form of example 6was repeated, with the duration of particle exposure after dissociationof the IgG from the SXC particles varied in increments to determine howmuch time was required to achieve the best results. Results wereessentially the same at 30 and 60 minutes, but inferior at lowerexposure times.

Example 8

An IgG-containing cell culture harvest containing 275,357 ppm host cellproteins, 5283 ppm DNA, and 13.96% aggregates was conditioned byaddition of 1% allantoin, then 0.025% ethacridine, then mixed for 15minutes. An equal mixture of MacroPrep High Q, MacroPrep High S,Macroprep tButyl, and Chelex-100 (Bio-Rad Laboratories), equilibrated inadvance by washing with 50 mM HEPES, 100 mM NaCl, pH 7.0. Equilibratedmixed particles were added to the impure IgG preparation in an amount of2% (v:v), then mixed overnight at 4-8 degrees C. Solids were removed bymicrofiltration. 1.25 mg of starch coated 200 nm magnetic particles wereadded to 20 mL of the conditioned impure IgG preparation. 20 mL of 36%PEG-6000 in 1.6 M NaCl, 50 mM HEPES, pH 7.0 was added gradually whilemixing on a vortex mixer at 500 rpm to produce a final concentration of18% PEG-6000 and 0.8 M NaCl. Vortex mixing was continued for 30 minutes,then the IgG-loaded particles were collected magnetically. TheIgG-loaded particles were washed with fresh 50 mM HEPES, 0.8 M NaCl, pH7.0, and the wash solution was removed. The wash buffer was removed andthe particles were washed again in the same manner. The wash buffer wasremoved, and the antibody was resolubilized in 50 mM HEPES, 1 M NaCl, pH7. A 1 mL column packed with an electropositive-hydrophobicchromatography medium (Capto adhere, GE Healthcare) and equilibrated tothe same conditions. The solubilized IgG was applied to the column,where the IgG bound and some contaminants were understood to have bound,while residual PEG bind. A 5 column volume wash with equilibrationbuffer was then applied to more thoroughly eliminate unbound componentsfrom the system. The IgG was eluted with a 10 column volume lineargradient ending at 50 mM HEPES, 300 mM NaCl, pH 7.0. Purificationperformance is indicated by the following Table, where post-conindicates post-conditions, post-NP indicates post nanoparticles, andpost-CA indicates post Capto adhere. The left-hand value under recoveryindicates recovery for that step, while the right-hand value indicatescumulative recovery for previous steps plus that step. bld indicatesbelow limit of detection. For more details refer to Gagnon et al 2014supra:

Step HCP (ppm) DNA(ppm) Aggregates (%) Recovery % Harvest 275,357 5,28313.96 100/100 Post-con. 91,275 9 4.88 98/98 Post-NP 441 bld 3.59 87/84Post-C 2 bld <0.05 81/69

Example 9

An IgG-containing cell culture harvest containing 176,244 ppm hostprotein contaminants and 19% aggregates was conditioned by addition of1% allantoin, 4% electropositive metal affinity particles (TREN 40 high,Bio-Works), mixed for 4 hours at room temperature. A sample removed foranalysis showed reduction of host proteins to 90,259 ppm, and aggregatesto 1.2%. The pH was reduced to 5.2, 0.5% caprylic acid was added, andthe mixture incubated for 2 hours. A sample removed for analysis showedhost proteins at 1,758 ppm and aggregates at about 0.4%. Solids wereremoved through an electropositive depth filter (Sartorius PC1). Hostproteins were reduced to 135 ppm and aggregates to less than 0.05%. Theantibody was purified by precipitation in 18% PEG-6000 at pH 7.0. Theprecipitate was then washed with 1.8 M ammonium sulphate to remove PEG,then the antibody was resolubilized in 50 mM Hepes, pH 7.0. Host proteinwas reduced to 32 ppm. After application to an anion exchangechromatography column (UNOsphere Q, Bio-Rad) operated in void exclusionmode at 50 mM Tris, pH 8.0, host protein was reduced to less than 1 ppm.A parallel experiment differing only in the PEG precipitation beingconducted in the presence of 800 mM NaCl reduced host protein to lessthan 1 ppm. The anion exchange step reduced host protein and aggregatesto an undetectable level.

Example 10

An IgG-containing cell culture harvest containing 286,010 ppm host.protein contaminants and 23% aggregates was conditioned by addition of1% allantoin and 0.025% ethacridine, and incubated stirring at roomtemperature for 1 hour. A 1:1:1 mixture of particles (Chelex-100,MacroPrep tButyl, Macroprep High Q, Bio-Rad) were mixed, equilibrated tophysiological conditions, and settled mixed particles were added to theharvest in a combined amount of 5%, then mixed for 2 hours at roomtemperature. Host protein was reduced to 43,058 ppm and aggregates to3.4%. In one series of experiments conducted at pH 8.0, the sample wasfractionated by precipitation with PEG-6000, in separate experimentswhere the concentration was 600 mM, 800 mM, 900 mM, and 1000 mM (1 M).The precipitates were then washed in PEG, 50 mM Tris, pH 8.0, afterwhich the antibody was resolubilized in 50 mM Tris, pH 8.0. Host proteinin that series was reduced to 51, 55, 45, and 41 ppm respectively. Anionexchange particles in the form of Dowex AG1X2 (Bio-Rad) were added toeach sample in an amount of 5% v/v and mixed for 60 minutes. Hostprotein across the series was reduced to 16, 17, 15, and 13 ppm. Anotherseries of experiments was run, identical in all details except theinitial PEG precipitation was performed at pH 7.0. Host protein afterthe PEG step was 44 ppm for the 600 mM NaCl track, 43 ppm for the 800 mMtrack, 29 ppm for the 900 mM track, and 31 ppm for the 1000 mM track.After Dowex treatment, host protein was reduced to 20, 17, 12, and 16ppm respectively.

Example 11

An IgG-containing cell culture harvest containing 286,010 ppm hostprotein contaminants and 23% aggregates was conditioned by addition of1% allantoin and 0.025% ethacridine, and incubated stirring at roomtemperature for 1 hour. A 1:1:1:1 mixture of particles (Chelex-100,MacroPrep tButyl, MacroPrep High Q from Bio-Rad), and electropositivemetal affinity particles (TREN 40 high from Bio-Works) were mixed,equilibrated to physiological conditions, and settled mixed particleswere added to the harvest in a combined amount of 5%, then mixed for 2hours at room temperature. Host protein was reduced to 38,061 ppm andaggregates to 1.4%. In a series of experiments conducted at pH 8.0, thesample was fractionated by precipitation with PEG-6000, in separateexperiments where the concentration of NaCl was 600 mM, 800 mM, 900 mM,and 1000 mM (1 M). The precipitates were then washed in PEG, 50 mM Tris,pH 8.0, after which the antibody was resolubilized in 50 mM Tris, pH8.0. Host protein was reduced to 79, 69, 56, and 57 ppm respectively.Anion exchange particles in the form of Dowex AG1X2 (Bio-Rad) were addedto each sample in an amount of 5% v/v and mixed for 60 minutes. Hostprotein across the series was reduced to 18, 17, 16, and 13 ppm. Anotherseries of experiments was run, identical in all details except theinitial PEG precipitation was performed at pH 7.0. Host protein afterthe PEG step was 94 ppm for the 600 mM NaCl track, 62 ppm for the 800 mMtrack, 67 ppm for the 900 mM track, and 46 ppm for the 1000 mM track.After Dowex treatment, host protein was reduced to 28, 9, 23, and 17 ppmrespectively.

Example 12

An IgM containing cell culture harvest containing 321,483 ppm hostprotein and 26% aggregates was conditioned by addition of 1% allantoin,0.025% ethacridine, and NaCl to produce a conductivity of 25 mS/cm. Themixture was incubated for 1 hour, solids were removed by centrifugation,and the liquid was flowed through a column packed with equal proportionsof MacroPrep tButyl, Macroprep High Q, Macroprep High S, and Chelex 100,where the volumetric ratio of column to harvest was 5%. Host protein wasreduced to 73,663 ppm and aggregates were reduced to 0.8%. The samplewas fractionated in parallel but separate experiments, both with 13%PEG-6000 at pH 7, one with 100 mM NaCl, the other with 800 mM NaCl. Theprecipitates were then washed with 13% PEG, 50 mM Hepes, pH 7.0 toremove the excess salt, then the IgM was resolubilized with 50 mM Hepes,pH 7.0. Host protein at 100 mM NaCl was reduced to 7,411 ppm. Hostprotein at 800 mM NaCl was reduced to 417 ppm. Aggregate contentincreased to 1.1%. The samples were applied to an anion exchangemonolith (CIM QA, BIA Separations) at pH 7.0 and eluted with a sodiumchloride gradient. Host proteins in the sample corresponding to PEGprecipitation at 100 mM NaCl were reduced to 1,424 ppm. Host proteins inthe sample corresponding to PEG precipitation at 800 mM NaCl werereduced to 63 ppm. Aggregates were less than 0.01% for bothpreparations.

The present methods disclosed herein may be combined with otherpurification methods to achieve higher levels of purification. Examplesof such other purification methods include, but are not limited to,other methods commonly used for purification of IgG, such as protein Aand other forms of affinity chromatography, anion exchangechromatography, cation exchange chromatography, hydrophobic interactionchromatography, immobilized metal affinity chromatography, andadditional mixed mode chromatography methods; also methods ofprecipitation, crystallization, and liquid-liquid extraction. It iswithin the purview of a person of ordinary skill in the art to developappropriate conditions for the various methods and integrate them withthe methods disclosed herein to achieve the necessary purification of aparticular antibody.

All references cited herein are incorporated by reference in theirentirety and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, chromatographyconditions, and so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the specification and attached claims are approximationsthat may vary depending upon the desired performance sought to beobtained by the present methods disclosed herein.

Many modifications and variations of this methods disclosed herein canbe made without departing from its spirit and scope, as will be apparentto those skilled in the art. The specific embodiments described hereinare offered by way of example only and are not meant to be limiting inany way. It is intended that the specification and examples beconsidered as exemplary only, with the true scope and spirit of themethods disclosed herein being indicated by the following claims.

What is claimed is:
 1. A method of purifying an antibody from apreparation comprising: (a) providing the preparation comprising anantibody; (b) adding allantoin to the preparation to a supersaturatingconcentration, wherein after adding the allantoin, the preparation issupersaturated with allantoin; (c) removing solids from the preparation;(d) contacting the preparation with polyethylene glycol (PEG) in anamount that is sufficient to cause the antibody to precipitate or adsorbon a nonionic surface, wherein the PEG has a nominal molecular weight ina range of 4,000 to 8,000 Daltons; and (e) adjusting a saltconcentration of the preparation before or during the contacting step,the adjusting step providing a sufficient salt concentration to producea conductivity between 50 mS/cm and 150 mS/cm.
 2. The method of claim 1,comprising, after the contacting, retaining the antibody on a porousmembrane, thereby allowing soluble contaminants to pass through themembrane.
 3. The method of claim 1, comprising, after the contacting,separating the antibody from soluble contaminants by sedimenting aprecipitate of the antibody by means of centrifugation.
 4. The method ofclaim 1, wherein the nonionic surface comprises one selected from thegroup consisting of a membrane, a monolith, particles, the particlesbeing optionally magnetic, and combinations thereof.
 5. The method ofclaim 1, wherein the method is carried out in a single integratedapparatus.
 6. The method of claim 1, wherein the nominal molecularweight of the PEG is in a range from 5,000 Daltons to 6,000 Daltons. 7.The method of claim 1, wherein the salt is selected from the groupconsisting of sodium chloride, potassium chloride, sodium acetate,potassium acetate, sodium thiocyanate, potassium thiocyanate,guanidinium hydrochloride, and combinations thereof.
 8. The method ofclaim 7, wherein the salt comprises sodium chloride adjusted to aconcentration in a range of 0.5 M to 1.5 M.
 9. The method of claim 1,further comprising adjusting a pH of the preparation, before or afterthe contacting, to a range of 5 to
 9. 10. The method of claim 1, whereinthe preparation is obtained from a biological source selected from thegroup consisting of a cell culture medium, an extract from culturedorganisms, and a bodily fluid.